1. Field of the Invention
The present invention relates to a multi-capillary electrophoretic apparatus employed for separation of protein or sequence determination for DNA.
2. Description of the Prior Art
A multi-capillary electrophoretic apparatus comprises a multi-capillary array electrophoresis part, an optical measuring part and a data processing part The multi-capillary array electrophoresis part has an arrangement of a plurality of capillary columns for injecting each of a plurality of samples into the capillary columns and simultaneously electrophoresing the same in all capillary columns. The optical measuring part irradiates the capillary columns with light in the multi-capillary array electrophoresis part, scans the irradiated positions perpendicularly to an electrophoresis direction, detects the intensity of light from the samples of the irradiated parts and measures scan waveforms. The data processing part produces time-series data as to each capillary column from the scan waveforms obtained by the optical measuring part.
A multi-capillary electrophoretic apparatus for sequence determination for DNA employs Sanger""s reaction and electrophoreses a DNA fragment sample prepared by labeling a primer or a terminator with a fluorescent material for detecting fluorescence from the DNA fragment sample in the course of electrophoresis and determining the base sequence.
A high-speed DNA sequencer having high sensitivity and high throughput is necessary for sequence determination for DNA such as a human genome having long base sequence. For example, a multi-capillary DNA sequencer having an arrangement of a plurality of capillary columns charged with gels is proposed in place of that employing flat slab gels. With such capillary columns, samples are not only easy to handle or inject but can also be electrophoresed at a high speed and detected in high sensitivity as compared with the slab gels. While bands are spread due to influence by Joulean heat or a temperature gradient is caused when a high voltage is applied to the slab gels, the capillary columns have no such problem and enable high-speed detection with small spreading of bands in high-speed electrophoresis with application of a high voltage.
Upon measuring scan waveforms by an optical measuring part in an online multi-capillary electrophoretic apparatus, a detected part of a capillary array 2 having an alignment of a plurality of capillary columns is scanned perpendicularly to a direction for elecbtrophoresing samples in the capillary columns as shown by a straight line 3 in FIG. 1 for receiving fluorescence from the samples passing through the scanned position. The fluorescence is received at for example 13000 points, in a single scanning. Each of these points is referred to as a sampling point or a data point The samples, which are DNA fragment samples, are labeled in four types in response to end bases adenine (A), guanine (G), thymine (T) and cytosine (C). Therefore, the base sequence can be determined by separating the received fluorescence into its spectral components thereby identifying the types of the bases passing through the scanned position.
Scan waveforms obtained by scanning the scan line 3 on a fixed time frame are detected as peaks by electrophoresing and passing the DNA fragment samples through columns C1, C2, C3, . . . , as shown in FIG. 2A. While symbols A, C, T and G denote DNA fragments of the four types of bases respectively, a common detector detects these four signals as light components of different wavelengths. Symbols t1, t2, . . . denote times of scanning the scan line 3 respectively at a rate of once a second, for example.
The DNA fragment samples pass through the position of the scan line 3 and hence the scan waveforms change with the elapse of time. When arranging data on a prescribed position of each column from the scan waveforms for the respective end bases as to each column, time-series data shown in FIG. 2B is obtained. FIG. 2B shows part of time-series data related to adenine (A) as to the column C1.
In the scan waveforms obtained by scanning the scan line 3, there may exist such signals exceeding input levels as those of adenine (A) and thymine (T) in the columns C2 and C3 in FIG. 2A. The input levels are determined by the detection range of the detector of the optical measuring part or the input range of an Axe2x80x94D converter capturing data in a data processing part When detected signals exceed the input levels, the peaks on the scan waveforms are saturated. When producing time-series data from the scan waveforms including the saturated peaks, the time-series data are distorted.
Also, the scan waveforms have tailing due to an electric time-constant of the detector. Therefore, in the scan waveform of a capillary column detected immediately after a capillary column allowing detection of strong fluorescence, influence by tailing of the strong signal appears with addition of signal intensity. This also results in distortion.
When producing time-series data from scan waveforms, positions for acquiring the time-series data from the scan waveforms are previously fixed with reference to the positions of the capillary columns. However, fluctuation of peak positions may be observed also in scan waveforms in a short time so assumed that the capillary column positions hardly fluctuate. When acquiring the time-series data from the scan waveforms on the basis of previously set capillary column position information in this case, it follows that the data are acquired around non-peak positions in the capillary columns depending on scanning, also resulting in distortion.
Furthermore, the positions of the capillary columns may fluctuate with the elapse of time. Also in this case, it follows that the data are acquired on positions varied with time in the capillary columns when acquiring the time-series data from the scan waveforms on the basis of previously set capillary column position information, also resulting in distortion.
When the time-series data are distorted due to tailing or fluctuation of the positions of data acquisition, errors may be caused when determining the base sequence.
Accordingly, an object of the present invention is to improve the precision of time-series data by reducing distortion caused when converting scan waveforms to time-series data.
A multi-capillary electrophoretic apparatus according to the present invention comprises a multi-capillary array electrophoresis part having an arrangement of a plurality of capillary columns for injecting each of a plurality of samples into the capillary columns and simultaneously electrophoresing the samples in all capillary columns, an optical measuring part for measuring scan waveforms by irradiating the capillary columns with light in the multi-capillary array electrophoresis part, scanning irradiated positions in a direction perpendicular to a electrophoresis direction, and detecting the intensity of light from the samples of irradiated parts, and a data processing part for producing time-series data as to each capillary column from the scan waveforms obtained by the optical measuring part
In order to reduce distortion caused in conversion to the time-series data, the data processing part comprises a saturated data correction part for correcting saturated peaks included in the scan waveforms, which are saturated beyond the detection range of a detector of the optical measuring part or the input range of an Axe2x80x94D converter capturing data in the data processing part, to light intensity value measured on the assumption that the peaks are unsaturated in an aspect of the present invention. The data processing part produces the time-series data on the basis of unsaturated scan waveform peaks and the light intensity value corrected by the saturated data correction part as to the saturated scan waveform peaks.
In order to correct saturated data, the data processing part preferably comprises a correction data storage part for storing correction data indicating the relationship between the number of data points of saturated parts of the saturated scan waveform peaks and light intensity data obtained on the assumption that the peaks are unsaturated. The saturated data correction part corrects measured light intensity value on the basis of the correction data stored in the correction data storage part as to the saturated peaks.
The correction data storage part can store correction data indicating the relationship between the number of the data points of the saturated parts and peak heights obtained on the assumption that the peaks are unsaturated. In this case, the saturated data correction part can employ the peak heights stored in the correction data storage part as time-series data of the saturated scan waveform peaks.
The correction data storage part can store correction data indicating the relationship between the number of the data points of the saturated parts and scan waveforms obtained on the assumption that the peaks are unsaturated. In this case, the saturated data correction part can employ the data on prescribed positions of the corrected scan waveforms of the capillary columns as time-series data of the saturated scan waveform peaks.
According to this aspect of the present invention, the saturated data correction part obtains the light intensity value measured on the assumption that the peaks are unsaturated for producing the time-series data with the corrected light intensity value, thereby suppressing distortion of the time-series data.
According to another aspect of the present invention, the data processing part comprises a tailing correction part for removing tailing components caused by electric time-constants of the light intensity signals of capillary columns located on front positions in the scan waveforms when producing the time-series data from the scan waveforms.
According to this aspect of the present invention, the time-series data can be obtained on the basis of correct signal intensity by removing influence by tailing.
According to yet another aspect of the present invention, the data processing part comprises a capillary position correction part for obtaining a position maximizing the light intensity signals around the previously set capillary column position for data acquisition, and acquires the time-series data on the basis of the position maximizing the light intensity signals obtained by the capillary position correction part when producing the time-series data from the scan waveforms.
According to a further aspect of the present invention, the data processing part has a function of periodically correcting the capillary column position information for data acquisition, and acquires the time-series data from the scan waveforms on the basis of the corrected capillary column position information when producing the time-series data from the scan waveforms.
Thus, distortion of the time-series data resulting from errors in the positions for acquiring the time-series data can be prevented by correcting the positions for acquiring the time-series data from the scan waveforms.
The multi-capillary electrophoretic apparatus may comprise any one or more than two of the saturated data correction part, the tailing correction part, the capillary position correction part having the function of obtaining the position maximizing the light intensity signal and the capillary position correction part having the function of periodically correcting the capillary column position information.
The correction data storage part can store the correction data indicating the relationship between the number of the data points of the saturated parts and the peak heights obtained on the assumption that the peaks are unsaturated so that the saturated data correction part can employ the peak heights stored in the correction data storage part as time-series data of the saturated scan waveform peaks.
The correction data storage part can also store correction data indicating the relationship between the number of the data points of the saturated parts and scan waveforms obtained on the assumption that the peaks are unsaturated so that the saturated data correction part can employ data on prescribed positions of corrected scan waveforms of the capillary columns as time-series data of the saturated scan waveform peaks.
The multi-capillary electrophoretic apparatus according to the present invention correcting the light intensity data of the saturated peaks included in the scan waveforms, removing the tailing components or correcting the capillary position information for acquiring data can suppress distortion of the time-series data.